Experiments and Analysis of Mosquito Flight Behaviors in a Wind Tunnel: An Introduction.

Mosquitoes detect and navigate to important resources, like a host, using combinations of different sensory stimuli. The relative importance of the sensory cues can change as the mosquito gets closer to their target. Other factors, both internal and external, can also influence the mosquito behavior. A mechanistic understanding of these sensory stimuli, and how they impact mosquito navigation, can now be readily studied using wind tunnels and associated computer vision systems. In this introduction, we present a behavioral paradigm using a wind tunnel for flight behavior analysis. The wind tunnel's large size with its associated cameras and software system for analysis of the mosquito flight tracks can be sophisticated and sometimes cost-prohibitive. Nevertheless, the wind tunnel's flexibility in allowing the testing of multimodal stimuli and scaling of environmental stimuli makes it possible to reproduce conditions from the field and test them in the laboratory, while also allowing the observation of natural flight kinematics.

Conducting an Analysis of Mosquito Flight Behaviors in a Wind Tunnel.

Prior to conducting wind tunnel experiments, mosquitoes must be prepared for testing. Important factors and state-dependent processes of the mosquito-like the sex, age, infection status, reproductive status, or nutritional status-should be evaluated and motivated by questions and hypotheses one seeks to address. Other critical external factors that can impact the mosquitoes' behavior and should be controlled for both in the colony and in the room where the wind tunnel experiments take place include the circadian rhythm, room temperature, light intensity, and relative humidity. Together, the internal and external factors, and wind tunnel design, ultimately control the behavior of the mosquito and, hence, the success of the experiments. In the present protocol, we describe methods using a standard wind tunnel design in which the fan pulls the air through the working section of the wind tunnel and the mosquito behavior is recorded by a multicamera system. Variations around the camera tracking system can be adapted according to the research questions being asked and include real-time tracking for both closed-loop and open-loop control of the stimulus environment or recording of the videos for off-line digitization and analysis. Within the working section, the sensory environment (odor, visual, wind) can be controlled to test the mosquito responses to different stimuli, and below we include different equipment and tools for modifying the stimuli the mosquito experiences during flight. Finally, the methods described here are applicable to multiple mosquito species, although the experiment parameters may need to be changed (e.g., ambient luminosity).

The olfactory gating of visual preferences to human skin and visible spectra in mosquitoes.

Mosquitoes track odors, locate hosts, and find mates visually. The color of a food resource, such as a flower or warm-blooded host, can be dominated by long wavelengths of the visible light spectrum (green to red for humans) and is likely important for object recognition and localization. However, little is known about the hues that attract mosquitoes or how odor affects mosquito visual search behaviors. We use a real-time 3D tracking system and wind tunnel that allows careful control of the olfactory and visual environment to quantify the behavior of more than 1.3 million mosquito trajectories. We find that CO2 induces a strong attraction to specific spectral bands, including those that humans perceive as cyan, orange, and red. Sensitivity to orange and red correlates with mosquitoes' strong attraction to the color spectrum of human skin, which is dominated by these wavelengths. The attraction is eliminated by filtering the orange and red bands from the skin color spectrum and by introducing mutations targeting specific long-wavelength opsins or CO2 detection. Collectively, our results show that odor is critical for mosquitoes' wavelength preferences and that the mosquito visual system is a promising target for inhibiting their attraction to human hosts.

Elimination of vision-guided target attraction in Aedes aegypti using CRISPR.

Blood-feeding insects, such as the mosquito, Aedes (Ae.) aegypti, use multiple senses to seek out and bite humans.1,2 Upon exposure to the odor of CO2, the attention of female mosquitoes to potential targets is greatly increased. Female mosquitoes are attracted to high-contrast visual cues and use skin olfactory cues to assist them in homing in on targets several meters away.3-9 Within close range, convective heat from skin and additional skin odors further assist the mosquitoes' evaluation as to whether the object of interest might be a host.10,11 Here, using CRISPR-Cas9, we mutated the gene encoding Op1, which is the most abundant of the five rhodopsins expressed in the eyes of Ae. aegypti. Using cage and wind-tunnel assays, we found that elimination of op1 did not impair CO2-induced target seeking. We then mutated op2, which encodes the rhodopsin most similar to Op1, and also found that there was no impact on this behavior. Rather, mutation of both op1 and op2 was required for abolishing vision-guided target attraction. In contrast, the double mutants exhibited normal phototaxis and odor-tracking responses. By measuring the walking optomotor response, we found that the double mutants still perceived optic flow. In further support of the conclusion that the double mutant is not blind, the animals retained an electrophysiological response to light, although it was diminished. This represents the first genetic perturbation of vision in mosquitoes and indicates that vision-guided target attraction by Ae. aegypti depends on two highly related rhodopsins.

Visuo-Motor Feedback Modulates Neural Activities in the Medulla of the Honeybee, Apis mellifera.

Behavioral and internal-state modulation of sensory processing has been described in several organisms. In insects, visual neurons in the optic lobe are modulated by locomotion, but the degree to which visual-motor feedback modulates these neurons remains unclear. Moreover, it also remains unknown whether self-generated and externally generated visual motion are processed differently. Here, we implemented a virtual reality system that allowed fine-scale control over visual stimulation in relation to animal motion, in combination with multichannel recording of neural activity in the medulla of a female honeybee (Apis mellifera). We found that this activity was modulated by locomotion, although, in most cases, only when the bee had behavioral control over the visual stimulus (i.e., in a closed-loop system). Moreover, closed-loop control modulated a third of the recorded neurons, and the application of octopamine (OA) evoked similar changes in neural responses that were observed in a closed loop. Additionally, in a subset of modulated neurons, fixation on a visual stimulus was preceded by an increase in firing rate. To further explore the relationship between neuromodulation and adaptive control of the visual environment of the bee, we modified motor gain sensitivity while locally injecting an OA receptor antagonist into the medulla. Whereas female honeybees were tuned to a motor gain of -2 to 2 (between the heading of the bee and its visual feedback), local disruption of the OA pathway in the medulla abolished this tuning, resulting in similar low levels of response across levels of motor gain. Our results show that behavioral control modulates neural activity in the medulla and ultimately impacts behavior.SIGNIFICANCE STATEMENT When moving, an animal generates the motion of the visual scene over its retina. We asked whether self-generated and externally generated optic flow are processed differently in the insect medulla. Our results show that closed-loop control of the visual stimulus modulates neural activity as early as the medulla and ultimately impacts behavior. Moreover, blocking octopaminergic modulation further disrupted object-tracking responses. Our results suggest that the medulla is an important site for context-dependent processing of visual information and that placing the animal in a closed-loop environment may be essential to understanding its visual cognition and processing.